CN104429944B - A kind of method that batch prepares sterile oil palm embryo sample - Google Patents
A kind of method that batch prepares sterile oil palm embryo sample Download PDFInfo
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- CN104429944B CN104429944B CN201410611726.9A CN201410611726A CN104429944B CN 104429944 B CN104429944 B CN 104429944B CN 201410611726 A CN201410611726 A CN 201410611726A CN 104429944 B CN104429944 B CN 104429944B
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Abstract
The invention discloses a kind of method that batch prepares sterile oil palm embryo sample.2/3rds are cut off along ripe oil palm fruit carpopodium center line with pruner, is chosen embryo with dissecting needle after breaking into two with one's hands；By the healthy and strong full embryo of outward appearance with after 0.01 0.05% mercury chloride flushing again in superclean bench with 0.01 0.05% the 30min of mercury chloride soaking disinfection 5；After the sterilized water rinsing of embryo after sterilization aqua sterilisa is removed with sterilizing filter paper, light culture 25 days under the conditions of 20 30 DEG C are seeded in the MS minimal mediums containing vitamin of PhytoTechnology companies, is transferred under the conditions of optical culture and is cultivated 15 25 days and can sprout.The present invention can obtain a large amount of sterile oil palm embryo samples in a short time, and pollution rate is low, and germination rate is high, disclosure satisfy that demand of the correlative study such as tissue cultures and the transcript profile based on embryo culture, protein group to a large amount of samples.
Description
Technical field
The present invention relates to the preparation of oil palm embryo sample, and in particular to a kind of batch prepares the side of sterile oil palm embryo sample
Method.
Background technology
Oil palm is important tropical oil-produced vegetable, mainly carries out seedling breeding by seed at present, but seed sprouting be present
The problems such as the cycle is long and germination rate is low.The physical limits such as the fine and close endosperm of seed hull and periphery of height lignifying are released, then
It is an important channel to carry out seedling breeding or seed sprouting correlative study by embryo culture, and obtaining complete sterile embryo is
Premise.Oil palm embryo is coniform in length, and length about 2-3mm, quality is soft crisp, is wrapped in hard fine and close endosperm, and periphery is successively
It is the seed hull of height lignifying and rich grease-contained pulp.Prior art usually requires band endosperm and sterilized, then super
Embryo is shelled in net workbench, takes that embryo efficiency is low and the integrality of embryo is poor, and the mass of 1000 kernel of embryo is only 1.2g or so, can not much be expired
Sufficient related experiment need to prepare the demand of a large amount of embryo samples to every batch.
It is to prepare the technical bottleneck of a large amount of embryo samples to take embryo efficiency.In view of oil palm embryo is tiny soft crisp, can only lead at present
Cross peel manually embryo.Oil palm endosperm containing embryo is only small-sized peanut grain size, subcircular and fine and close hard.During band endosperm stripping embryo,
Stress point is not easy to grasp, and especially in the superclean bench that operating space is limited, the machinery during embryo is easily taken because of craft
Damage causes subsequent contamination or can not normally sprouted, and therefore seriously drags and take embryo speed and inoculation speed slowly.It is and straight with pruner
Connect taken out from oil palm fruit naked embryo have the advantages that lifting surface area it is big, it is workable, take that embryo speed is fast, embryo integrality is good.
In addition, directly carrying out naked embryo sterilization has the advantages that sterilization reagent concentration is low, disinfecting time is short, environmental pollution is small, it is to prepare greatly
The Perfected process of gauging palm fibre embryo sample.
The content of the invention
The shortcomings that it is an object of the invention to overcome prior art and deficiency, there is provided a kind of batch prepares sterile oil palm embryo
The method of sample, this method can obtain a large amount of sterile oil palm embryo samples in a short time, disclosure satisfy that tissue cultures and
Demand of the correlative studys such as the transcript profile based on embryo culture, protein group to a large amount of samples.
The purpose of the present invention is achieved through the following technical solutions：
A kind of method that batch prepares sterile oil palm embryo sample, comprises the following steps：
(1) ripe oil palm fruit is collected, 2/3rds are cut off along carpopodium center line with pruner, is broken into two with one's hands i.e. it can be seen that breast
The embryo of white, is then chosen embryo with dissecting needle.
(2) selected with tweezers and retain the healthy and strong full embryo of outward appearance.
(3) chlorination of again in superclean bench using 0.01-0.05% is rinsed after embryo with 0.01-0.05% mercury chloride
Mercury soaking disinfection 5-30min.
(4) embryo obtained in (3) is rinsed to the mercury chloride for removing residual with aqua sterilisa, then is removed and sterilized with sterilizing filter paper
Water, light culture 2-5 days under the conditions of 20-30 DEG C are seeded in MS minimal mediums, transfer under the conditions of optical culture and cultivate 15-
It can sprout within 25 days；Described MS minimal mediums are the MS minimal mediums containing vitamin of PhytoTechnology companies.
Optical culture condition described in step (4) is preferably：Intensity of illumination is 1500-3500LX, temperature is 25-35 DEG C.
Preferably, described batch prepares the method for sterile oil palm embryo sample and comprised the following steps：
(1) ripe oil palm fruit is collected, 2/3rds are cut off along carpopodium center line with pruner, is broken into two with one's hands i.e. it can be seen that breast
The embryo of white, then embryo is chosen, be transferred to and be fixed in the Polyester fibre gauze of beaker mouth with dissecting needle.
(2) basis, which visually observes, compares, and is selected with tweezers and retains the healthy and strong full embryo of outward appearance.
(3) spray with the watering can equipped with 0.01-0.05% mercury chloride on gauze flushing embryo, then medication spoon is by embryo
It is transferred in the sterilizing beaker in superclean bench, with 0.01-0.05% mercury chloride soaking disinfection 5-30min.
(4) embryo obtained in (3) is rinsed into the 3-5 mercury chloride for removing residual with aqua sterilisa, is transferred to and is lined with sterilizing
Aqua sterilisa is removed in the sterilizing culture dish of filter paper, is then seeded to the MS containing vitamin equipped with PhytoTechnology companies
In minimal medium culture dish；First light culture 2-5 days under the conditions of 20-30 DEG C of culture dish being inoculated with, transfer to optical culture
Under the conditions of cultivate 15-25 days and can sprout.
The present invention is had the following advantages that relative to prior art and effect：
(1) embryo is directly taken with pruner and dissecting needle, it is workable, take embryo speed fast, embryo efficiency is manually taken up to 100
Individual/hour, and embryo integrality is good.
(2) due to the difference of oil palm fruit ear developmental condition and fruit adnation position, the developmental condition and turgor of embryo
Also it is different, and hypogenetic embryo easily parasitic various fungies and bacterium, and be not easy to sprout or late growing stage is bad.This hair
The hypogenetic embryo of bright rejected in advance, reduces embryo pollution rate, while improve follow-up germination rate.
(3) oil palm embryo quality is tender, to the poor resistance of the common disinfectantses such as alcohol, sodium hypochlorite, mercury chloride, such as disappears
Poison is dealt with improperly, easily leads to not sprout because sterilization dose is excessive, and sterilization dose deficiency can cause largely to pollute.This hair
The bright mercury chloride with 0.01-0.05% sterilizes 5-30min, and disinfection concentration is low, the time is short, and environmental pollution is small, can be by the whole of embryo
Body pollution rate is controlled within 5%, and does not influence to sprout, and greatly reduces the workload of follow-up embryo culture.
(4) culture medium of selection contains vitamin, has certain restoration and protection effect to the naked embryo after sterilization, helps simultaneously
In the brown stain probability for reducing embryo.Condition of culture simulates the pattern that embryo is sprouted in endosperm, first dark under the conditions of 20-30 DEG C
Culture 2-5 days, embryo is set to transfer to further activation embryo germination under the conditions of optical culture by the abundant water swelling of imbitition
Process, embryo pollution rate is low, speed of germinating is fast, can prepare sterile oil palm embryo sample in batches and be used for tissue cultures and based on embryo
The correlative studys such as the transcript profile of culture, protein group.
Embodiment
Further detailed description is done to the present invention with reference to specific embodiment.It should be understood that the following examples are only used
In the explanation present invention rather than limitation the scope of the present invention.
The MS minimal mediums used in following embodiments are that MS of the PhytoTechnology companies containing vitamin is trained substantially
Support base.The MS minimal mediums of PhytoTechnology companies are divided to two kinds, and one kind contains vitamin, and one kind is free of vitamin.
Embodiment 1
(1) ripe oil palm fruit is collected, 2/3rds are cut off along carpopodium center line with pruner, is broken into two with one's hands i.e. it can be seen that breast
The embryo of white, is then chosen from by embryo with dissecting needle, is transferred to and is fixed in the Polyester fibre gauze of beaker mouth.
(2) compares according to visually observing, chosen to remove with pincet short and small, thin and weak, shrivelled etc. has notable difference with other embryos
Embryo, only retain the healthy and strong full embryo of outward appearance.
(3) sprayed with the watering can equipped with 0.01% mercury chloride on gauze and rinse the embryo that may be stained with pollutant, then
Clean embryo is transferred in the sterilizing small beaker in superclean bench with small medicine spoon, with 0.01% mercury chloride soaking disinfection
30min。
(4) embryo of acquisition is rinsed into 3 mercury chloride for removing residual with sterile purified water, is transferred to and is lined with sterilizing filter paper
Sterilizing culture dish in remove unnecessary sterile purified water, be then seeded to ready PhytoTechnology companies in advance
In MS minimal mediums containing vitamin.First light culture 2 days under the conditions of 25 DEG C of culture dish being inoculated with, transfer to optical culture
Being cultivated 15 days under the conditions of (intensity of illumination 1500-3500LX, temperature are 25 DEG C) to sprout, and pollution rate 4.6%, finally sprout
Hair rate is 85.2%.
Embodiment 2
(1) ripe oil palm fruit is collected, 2/3rds are cut off along carpopodium center line with pruner, is broken into two with one's hands i.e. it can be seen that breast
The embryo of white, then embryo is chosen, be transferred to and be fixed in the Polyester fibre gauze of beaker mouth with dissecting needle.
(2) compares according to visually observing, chosen to remove with pincet short and small, thin and weak, shrivelled etc. has notable difference with other embryos
Embryo, only retain the healthy and strong full embryo of outward appearance.
(3) sprayed with the watering can equipped with 0.01% mercury chloride on gauze and rinse the embryo that may be stained with pollutant, then
Clean embryo is transferred in the sterilizing small beaker in superclean bench with small medicine spoon, with 0.05% mercury chloride soaking disinfection
5min。
(4) embryo of acquisition is rinsed into 5 mercury chloride for removing residual with sterile purified water, is transferred to and is lined with sterilizing filter paper
Sterilizing culture dish in remove unnecessary sterile purified water, be then seeded to ready PhytoTechnology companies in advance
In MS minimal mediums containing vitamin.First light culture 3 days under the conditions of 25 DEG C of culture dish being inoculated with, transfer to optical culture
Being cultivated 20 days under the conditions of (intensity of illumination 1500-3500LX, temperature are 30 DEG C) to sprout, and pollution rate 3.9%, finally sprout
Hair rate is 81.4%.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (1)
1. a kind of method that batch prepares sterile oil palm embryo sample, it is characterised in that comprise the following steps：
(1) ripe oil palm fruit is collected, 2/3rds are cut off along carpopodium center line with pruner, is broken into two with one's hands i.e. it can be seen that milky
Embryo, then embryo is chosen with dissecting needle, is transferred to and is fixed in the Polyester fibre gauze of beaker mouth；
(2) basis, which visually observes, compares, and is chosen with pincet and removes short and small, thin and weak, the shrivelled embryo for having notable difference with other embryos,
Only retain the healthy and strong full embryo of outward appearance；
(3) sprayed with the watering can equipped with 0.01% mercury chloride on gauze and rinse the embryo that may be stained with pollutant, then with small
Clean embryo is transferred in the sterilizing small beaker in superclean bench by medicine spoon, with 0.05% mercury chloride soaking disinfection
5min；
(4) embryo of acquisition is rinsed into 5 mercury chloride for removing residual with sterile purified water, is transferred to and is lined with going out for sterilizing filter paper
Unnecessary sterile purified water is removed in bacterium culture dish, ready PhytoTechnology companies in advance is then seeded to and contains dimension
In the MS minimal mediums of raw element；First light culture 3 days under the conditions of 25 DEG C of culture dish being inoculated with, transfer to optical culture condition
Lower culture 20 days；
The condition of the optical culture be intensity of illumination be 1500-3500LX, temperature be 30 DEG C.
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| CN101965798B (en) * | 2010-10-26 | 2012-08-22 | 青岛农业大学 | Peanut somatic embryo induction and plant regeneration method |
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